Kinase anchor protein muteins, peptides thereof and related documents
First Claim
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1. A polypeptide that is a mutein of a D-AKAP2 polypeptide, wherein the mutein exhibits modified binding to a regulatory subunit of PKA compared to a native D-AKAP2.
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Abstract
A-kinase anchor protein (AKAPs) muteins, peptides thereof, and nucleic acids encoding the peptides are provided herein. Also provided are transgenic animals, cells comprising transgenes and various methods employing such peptides.
213 Citations
106 Claims
- 1. A polypeptide that is a mutein of a D-AKAP2 polypeptide, wherein the mutein exhibits modified binding to a regulatory subunit of PKA compared to a native D-AKAP2.
- 3. A polypeptide that is a mutein of a D-AKAP2 polypeptide, wherein the mutein exhibits modified binding to a regulatory subunit of PKA compared to a control.
- 15. A peptide comprising between 15 and 39 amino acid residues, wherein the peptide comprises amino acids 7-21 of SEQ ID NOs:
-
27. A peptide having substantially no ability to bind to PKA-RIα
- subunit while maintaining the ability to bind to PKA-RIIα
subunit, compared to the peptide of SEQ ID NOs;
1 or 2, wherein the peptide is selected from the group consisting of;
- subunit while maintaining the ability to bind to PKA-RIIα
-
28. A peptide having substantially no ability to bind to a PKA-RIα
- subunit while maintaining a reduced ability to bind to a PKA-RIIα
subunit, compared to the peptide of SEQ ID NOs;
1 or 2, wherein the peptide is selected from the group consisting of;
- subunit while maintaining a reduced ability to bind to a PKA-RIIα
-
29. A peptide that has enhanced binding to both PKA-RIα
- subunit and PKA RIIα
subunit, compared to the peptide of SEQ ID NOs;
1 or 2, wherein the peptide is selected from the group consisting of;
- subunit and PKA RIIα
-
30. A peptide that has enhanced ability to bind to PKA-RIα
- subunit, while maintaining a normal or reduced ability to bind to PKA-RIIα
subunit, compared to the peptide of SEQ ID NOs;
1 or 2, wherein the peptide is selected from the group consisting of;
- subunit, while maintaining a normal or reduced ability to bind to PKA-RIIα
-
31. A peptide that has an ability to bind to PKA-RIα
- subunit but substantially no ability to bind to PKA-RIIα
subunit, compared to the peptide of SEQ ID NOs;
1 or 2, wherein the peptide comprises
- subunit but substantially no ability to bind to PKA-RIIα
-
32. A peptide that has an enhanced ability to bind to PKA-RIα
- subunit, and a reduced ability to bind to PKA-RIIα
subunit, compared to the peptide of SEQ ID NOs;
1 or 2, wherein the peptide has a D-amino acid at the position indicated in bold and wherein the peptide is selected from the group consisting of;
- subunit, and a reduced ability to bind to PKA-RIIα
-
33. A peptide that has enhanced ability to bind to PKA-RIα
- subunit, and a reduced ability to bind to PKA-RIIα
subunit, compared to the peptide of SEQ ID NOs;
1 or 2, wherein the peptide is selected from the group consisting of;
- subunit, and a reduced ability to bind to PKA-RIIα
-
34. A peptide that has enhanced ability to bind to PKA-RIα
- subunit, while maintaining a normal ability to bind to PKA-RIIα
subunit, compared to the peptide of SEQ ID NOs;
1 or 2, wherein the peptide is;
- subunit, while maintaining a normal ability to bind to PKA-RIIα
- 38. A transgenic non-human knock-in animal comprising a gene encoding an animal homolog of human D-AKAP2, wherein the gene has been modified to encode one, two or more amino acid substitutions in a 27-mer PKA binding region of the animal homolog of human D-AKAP2 corresponding to amino acids 623-649 of human D-AKAP2 set forth in SEQ ID NOs:
-
51. A cell, comprising heterologous nucleic acid that encodes a mammalian D-AKAP2 variant protein or portion that exhibits a preferred or exclusive binding to PKA-RIα
- subunits relative to PKA-RIIα
subunits;
or enhanced binding to both RIα and
RIIα
subunits, compared to the unmodified full length D-AKAP2 protein, wherein the D-AKAP2 variant protein or portion thereof comprises at least one single amino acid substitution in the mammalian D-AKAP2 selected from the group consisting of substitutions that correspond to the substitution in SEQ ID NO;
63, 64 or 65 of one, two or more;
of Q at residue 631 with F, I, L, V, H, M, R, T, W or Y;
of L at residue 634 with F, W or Y;
of V at residue 643 with I, L or W; and
of M at residue 647 with F, I, L, T, V, W or Y.
- subunits relative to PKA-RIIα
-
52. A cell, comprising heterologous nucleic acid that encodes a mammalian D-AKAP2 variant protein or portion that exhibits a preferred or exclusive binding to PKA-RIIα
- subunits relative to PKA-RIα
subunits, compared to the unmodified full length D-AKAP2 protein, wherein the D-AKAP2 variant protein or portion thereof comprises at least one single amino acid substitution in the mammalian D-AKAP2 selected from the group consisting of substitutions that correspond to the substitution in SEQ ID NO;
63, 64 or 65 of one, two or more;
of L at residue 634 with A, C, or K;
of A at residue 635 with F, H, I, K, L, M, N or S;
of W at residue 636 with C;
of K at residue 637 with C;
of K at residue 640 with C;
of M at residue 641 with C;
of S at residue 644 with C; and
of D at residue 645 with C.
- subunits relative to PKA-RIα
-
55. A method for modulating the amount of PKA-RIα
- bound to D-AKAP2 in a cell comprising;
changing the effective intracellular concentration of the Val(646) isoform of D-AKAP2 corresponding to SEQ ID NO;
65 in the cell. - View Dependent Claims (56, 57)
- bound to D-AKAP2 in a cell comprising;
-
58. A method for altering the intracellular location of PKA in a cell comprising:
changing the effective intracellular concentration of the Val(646) isoform of D-AKAP2 corresponding to SEQ ID NO;
65 in the cell.- View Dependent Claims (59, 60)
-
62. A method of increasing the longevity of a subject in need thereof, comprising identifying a subject having the 646(Val) isoform of D-AKAP2 therein, and treating the subject with an agent that disrupts the binding the 646(Val) isoform of D-AKAP2 to RIα
- subunit of PKA.
- View Dependent Claims (63)
-
64. A method for altering the ratio of PKA-RIα
- /PKA-RIIα
bound to D-AKAP2 in a cell comprising;
increasing the concentration of a Val(646) variant of D-AKAP2 corresponding to SEQ ID NO;
65 in a cell, thereby increasing the ratio of PKA-RIα
PKA/PKA-RIIα
bound to D-AKAP2 in a cell.
- /PKA-RIIα
-
65. A method of decreasing the concentration of cAMP required to stimulate a cAMP mediated signalling pathway, comprising:
increasing the concentration of a Val(646) variant of D-AKAP2 corresponding to SEQ ID NO;
65 in a cell.
-
66. A method of screening for agents that decrease the binding of a Val(646) variant of D-AKAP2 with RIα
- PKA, comprising;
combining a candidate agent with a cell comprising a nucleotide sequence which encodes a Val(646) variant D-AKAP protein corresponding to SEQ ID NO;
65, operably linked to a promoter such that the nucleotide sequence is expressed as a D-AKAP2 protein in the cell; and
determining the effect of the agent upon the localization of PKA to the mitochondria, wherein a decrease in localization to the mitochondria identifies an agent that decreases the binding of a Val(646) variant of D-AKAP2 with RIα
PKA.
- PKA, comprising;
-
67. A method of screening for agents that decrease the binding of a Val(646) variant of D-AKAP2 to an RIα
- subunit of PKA, comprising;
combining a candidate agent with an admixture comprising RIα and
a D-AKAP2 peptide sequence that binds to RIα
; and
determining the effect of the agent upon the localization of PKA to a mitochondria. - View Dependent Claims (68, 69, 70)
- subunit of PKA, comprising;
-
86. A peptide, comprising between 12 and 39 amino acid residues, wherein the peptide comprises a segment of SEQ ID NOs:
- 1 or 2, wherein the length of the segment ranges from at least amino acids 12-23 of SEQ ID NOs;
1 or 2 up to amino acids 1-27 of SEQ ID NOs;
1 or 2. - View Dependent Claims (87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103)
- 1 or 2, wherein the length of the segment ranges from at least amino acids 12-23 of SEQ ID NOs;
-
104. A peptide selected from SEQ ID NOs:
- 3-9.
Specification